Obtainment of a rough-type Salmonella enteritidis and its genetic modifications for use as an avian vaccine

ABSTRACT

The present invention relates to a strain of Salmonella enteritidis 3934vac, which has been deleted the waaL gene to obtain a rough phenotype (3934vac DwaaL), the obtaining procedure and the oligos used with the objective of reducing toxicity and maintaining immunogenicity for its application as a vaccine. Another aspect of the present invention relates to a strain of Salmonella enteritidis 3934vac DwaaL, i.e. rough type, which has been modified to express the gene of the avian adenovirus type I fiber, in addition to the procedure for obtaining a Salmonella enteritidis 3034 vac DwaaL strain expressing an AvA-I fiber gene. The invention also comprises the development of a new, live, recombinant, effective and innocuous avian vaccine against the AvA-I virus developed via an insertion and integration process of AvA-I fiber genes in the chromosome of an attenuated and non-pathogenic strain of the bacterium Salmonella enteritidis.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a national stage application under 35 U.S.C. 371 andclaims the benefit of PCT Application No. PCT/PE2018/000014 having aninternational filing date of 12 Jun. 2018, which designated the UnitedStates, which PCT application claimed the benefit of Peru ApplicationNo. 000992-2017/DIN filed 12 Jun. 2017, the disclosure of each of whichare incorporated herein by reference.

REFERENCE TO SEQUENCE LISTING

This application contains a Sequence Listing submitted as an electronictext file named “20210617-SeqList.txt”, having a size in bytes of 63 KB,and created on 17 Jun. 2021. The information contained in thiselectronic file is hereby incorporated by reference in its entiretypursuant to 37 CFR § 1.52(e)(5).

SECTOR OF THE TECHNIQUE

The present invention falls within the field of Animal Health. Morespecifically, the invention relates to the development of a strain ofattenuated and rough-phenotype Salmonella enterica serovar Enteritidis(Salmonella enteritidis or SE), obtained by deletion of a gene involvedin the synthesis of lipopolysaccharide. In this context, it alsoincludes the use of this strain as a vaccine vector against the AvA-Ivirus (Aviadenovirus serogroup 1), via a process of insertion andintegration into its chromosome of the genes that encode the AvA-I fiberantigen. The SE vector that is used has been genetically modified tofunction as a vehicle for the expression of the immunodominant genes ofAvA-I virus fiber, with the goal of stimulating an effective and lastingimmune response against HCI (viral hepatitis by body inclusion) inbirds. The strain generated is completely safe because it is free of thetwelve genes encoding the proteins of the signaling pathway of thesecondary cyclic di-GMP messenger, the sigma RpoS factor and the WaaLprotein. Therefore, this strain is proposed as a new effective and saferecombinant live vaccine against the AvA-I virus, suitable to vaccinateavian populations.

All the components of this vaccine have been specifically designed anddeveloped with the three successive priority factors of efficacy, safetyand costs.

BACKGROUND OF THE INVENTION

A growth of 30% is expected in the poultry sector for the next 8 to 10years; this growth is accompanied by successful health programs, whichin turn depend on effective and safe vaccines. HCI/SHP (inclusion bodyhepatitis/hepatitis hydropericardium syndrome) is a disease that causesup to 50% mortality in affected birds and is a latent problem in Peru,because despite the vaccination there are still outbreaks, a problemthat is not solved because the technology used for the elaboration ofvaccines is the same for 20 years.

Recently, the techniques of genetic manipulation, added to theavailability of bacterial genomes, the better knowledge of themechanisms of pathogenesis and the immune response have allowed newapproaches for the development of attenuated bacterial strains asvectors of heterologous genes. Thus, enteropathogenic bacteria, such asListeria monocytogenes, Salmonella spp, Shigella spp, Vibrio cholerae,Yersinia enterocolitica or Bordetella pertussis have been used aseffective vectors as vaccines. Of all of them, Salmonella spp has giventhe most promising results. Thus, vaccine vectors based on attenuatedSalmonella typhimurium strains have been used to generate a protectiveimmune response against viral, bacterial and protozoan pathogens and asvehicle to carry anti-cancer treatments. In the specific case of birds,this type of vaccine has been evaluated as a gene vector for infectiousbronchitis virus, avian influenza virus, avian reovirus and Clostridiumperfringens. All these works agree with the fact that these vaccineswere able to provoke a lasting protective immune response, besides beingsafe to avoid the risk of causing the disease. Considering the aspectsmentioned above, there is a great support in the success that thedevelopment of a recombinant live vaccine against AvA-I using as avector an attenuated SE strain would offer the poultry industry. Theseadvantages can be summarized as follows: (1) severe attenuation throughseveral processes of directed mutagenesis through a mechanism of allelicexchange that prevents reversion, do not introduce exogenous DNA andconvert the resulting strain into a totally safe vaccine vector; (2) theamount of expression of the AvA-I antigen can be regulated by geneticmanipulation; (3) it can be administered by oral inoculation; (4) itstrongly stimulates the innate and adaptive immune system; and, (5) inturn, it would provide protection against SE. Although the developmentof vaccines using bacterial vectors has been and is currently the objectof numerous investigations, to date there are no licensed commercialvaccines based on SE. Therefore, the development of this vaccine wouldset a good precedent in this area of research and development and wouldnot have competition in national and international market.

The strain used in the present invention was isolated and described bySolano, C., Garcia, B. Valle, J., Berasain, C. Chigo J. M., Gamazo, C.,& Lasa, I. Genetic Analysis of Salmonella enteritidis biofilm formation:Critical role of cellulose. Molecular Microbiology, 43(3), pp. 793-808.This study reports a new screening method based on the fluorescence ofcolonies on calcofluor agar plates to identify mutants of Salmonellaenteritidis that are defective in biofilm formation. These results notonly confirmed the requirement of genes already described for themodulation of multicellular behavior in Salmonella typhimurium and otherspecies but also revealed new aspects of the biofilm formation process,such as new genetic elements named as bcsABZC and bcsEFG operons,necessary for the synthesis of an exopolysaccharide hydrolysable bycellulase. Non-polar mutations of the BcsC and bcsE genes, as well ascomplementation experiments, demonstrated that both operons areessential for cellulose biosynthesis in the LB and ATM media, both in S.enteritidis and S. typhimurium. This study also showed that the biofilmproduced by S. enteritidis is composed of different components,suggesting that the composition and regulation of the biofilm depends onenvironmental conditions. In addition, the results suggest thatcellulose is not involved in the virulence of S. enteritidis, althoughmutants deficient in the production of this polysaccharide were moresensitive to chlorine treatments, suggesting that cellulose productionand the formation of biofilm would be an important factor in thesurvival of S. enteritidis on surfaces. In a later study, Solano C.,Garcia, B., Latasa, C. Toledo-Arana, A. Zorraquino, V. Valle, JoanCasals, Enrique Pedroso and Iñigo Lasa. Genetic Reductionist Approachfor Dissecting Individual roles of GGDEF proteins within the c-di-GMPsignaling network in Salmonella. Proceedings of the National Academy ofSciences of the United States of America, 106 (19, pp. 7997-8002. (2009)performed an ambitious genetic approach in which the 12 genes thatencode proteins with enzymatic activity of diguanylate cyclase (DGC,proteins with GGDEF domain) were deleted in the same strain describedabove. These enzymes synthesize the secondary messenger c-di-GMP, amolecule whose involvement in key bacterial biological processes such asthe formation of biofilms or virulence has been widely studied innumerous species. In the specific case of this strain of SE, the totaldepletion of c-di-GMP correlates with the loss of the capacity to formbiofilms, a slight attenuation in a murine model and a greatersensitivity to extreme environmental conditions.

As a result of the genetic approach described above, patent document ES2324084B1 describes the invention of a method that allows producingmultiple modifications in the chromosome of Gram negative bacteria andstrains of Salmonella deficient in synthesis of C-di-GMP obtained by it.Said invention also relates to two mutant strains of enteric Salmonellaobtained by said method in which the twelve genes encoding proteins withGGDEF domain have been deleted (mutant ΔXII::Km and mutant ΔXII::Clo)and to the use thereof as expression vectors, immunotherapeutic agentsand in metabolic studies.

Garcia Ona Enrique. “Análisis de una cepa avirulenta de Salmonellaenteriditis para su use como vacuna viva atenuada. Universidad Públicade Navarra (2011), the Thesis: Analysis of an Avirulent Strain ofSalmonella enteritidis for Use as Live Attenuated Vaccine, studied thepotential of several derived strains included in document ES 2324084B1for the development of a vaccine able to induce an immune response,decrease the colonization of organs and the release of Salmonella infeces in intensive pig farms. This work concluded that the candidateSalmonella strain for the development of a vaccine would be the strainknown as S. Enteritidis 3934ΔXII*, which has deleted the 12 genes thatencode proteins with GGDEF domain and carries an additional deletion ina chromosomal region that includes the transcriptional regulator RpoS.This strain is avirulent, has a diminished ability to survive in theenvironment and since it does not contain exogenous DNA, it would not beclassified as GMO (genetically modified organism). Oral immunizationassays using a murine infection model, revealed that immunization with asingle dose of 10⁷ ufc (Colony Forming Units) of this strain is capableof inducing a protective immune response against Salmonella infection,indicating that strain may be a suitable candidate for use as a liveattenuated vaccine.

Latasa, C., Echeverz, M., Garcia, B., Gil, C., Garcia-ona, E., Burgui,S. Noelita Caseres, S. Hervás-Stubss., J. J., Lasarte, I. Lasarte., C.Solano Evaluation of Salmonella strain lacking the secondary messengerc-di-GMP and Rpos as a live oral vaccine. PLos ONE, 11(8), pp. 1-22(2016) conducted the following study with the objective of constructinga new live attenuated Salmonella vaccine: first, the impact of theabsence of cyclic di-GMP (c-di-GMP) on the Salmonella virulence wasanalyzed. As previously mentioned, C-di-GMP is a secondary intracellularmessenger that controls a wide range of bacterial processes, includingthe formation of biofilm and the synthesis of virulence factors, as wellas modulating the innate immune response of the host through the STINGreceptors. The results described in this article showed that a multiplemutant of Salmonella, from whose genome the twelve genes encodingdiguanylate cyclase proteins have been deleted and, as a consequence,cannot synthesize c-di-GMP, presents a moderate attenuation in a modelof systemic murine infection. A further controlled mutation (unlike theS. enteritidis 3934ΔXII* strain, in which the loss of this sigma factorhad resulted from a natural counter-selection) of the rpoS gene resultedin a synergistic attenuating effect, which led to a highly attenuatedstrain, called ΔXIII. This strain is sufficiently immunogenic to protectmice against an oral lethal attack of a virulent strain of S.typhimurium. The immunogenicity of ΔXIII depends both on the activationof the humoral and cellular response and is characterized by theproduction of opsonizing antibodies and the induction of significantlevels of IFN-γ, TNF-A, IL-2, IL-17 and IL-10. ΔXIII is capable offorming biofilms and does not survive under desiccation conditions,which indicates that it could be easily removed from the environment. Inaddition, ΔXIII has DIVA (Differentiating Infected from VaccinatedAnimals) properties, which allow the differentiation of infected andvaccinated animals. In order to facilitate the terminology, the ΔXIIIstrain is also known as 3934vac.

Additionally, the paper of Kong Q., Yang, J., Liu, Q., Alamuri, P.,Roland, K. L. & Curtiss, R. Effect of Deletion of Genes Involved inLipopolysaccharide Core and O-antigen synthesis on virulence andImmunogenicity of Salmonella enterica serovar Typhimurium. Infection andImmunity, 79(10), pp. 4227-4239. (2011), describes the effect of theelimination of the O-antigen and the sugars of the core in the virulenceand immunogenicity of Salmonella enterica serotype typhimurium. In thisstudy, non-polar mutations by deletion were performed in the waaG(rfaG), waaL (rfaI), rfaH, waaJ (rfaJ), wbaP (rfbP), waaL (rfaL), or wzy(rfc) genes in a wild-type strain of S. typhimurium. After confirmingthe structure of the LPS, several properties in vitro and in vivo ofeach mutant were analyzed and all mutants showed a significantattenuation and lower invasion capacity compared to the wild-typeparental strains when administered to BALB/c mice orally. In addition,the strains carrying a mutation in waaG and waaL were deficient in thecolonization of the Peyer patches and liver, this deficiency beingpartially compensated after the intranasal administration of the mutantDwaaL. In the context of an attenuated vaccine strain supplying thepneumococcal antigen PspA, all mutations tested resulted in reducedimmune responses against PspA and Salmonella antigens. These resultsindicate that non-reversible truncation of the outer core is not aviable option to develop a live oral Salmonella vaccine, whereas a wzymutant, which retains one unit of the O-antigen, would be adequate tostimulate optimal protective immunity to homologous or heterologousantigens orally, intranasally or intraperitoneally.

GENERAL DESCRIPTION OF THE INVENTION

The present invention relates to an attenuated Salmonella enteritidisstrain that has been modified by genetic engineering techniques todelete the waaL gene and therefore has a rough phenotype. Another aspectof the present invention relates to said mutant Salmonella enteritidisstrain in the waaL gene that also carries the genes encoding the AvA-Ifiber antigen on its chromosome. This strain, apart from having a roughphenotype and being avirulent, confers protection against AvA-I.Additionally, the invention comprises the process for obtaining anattenuated Salmonella enteritidis strain with a rough phenotype andcarrying out the integration of the genes encoding the AvA-I fiber intoits chromosome, including the plasmids and methods used to obtain saidstrain.

With the aim of reducing the toxicity and maintaining the immunogenicityof the strains based on the 3934vac genetic background (SE ΔXIII), oneaspect of the present invention relates to the deletion of the waaLgene. The mutation of this gene results in a rough phenotype because thelipopolysaccharide is composed only of the core and lipid A, having lostthe ability to synthesize the O-antigen. This mutation has theadditional advantage of allowing the differentiation of animalsvaccinated and naturally colonized by wild-type strains of Salmonellaenteritidis and typhimurium.

For the generation of the rough mutants, an experimental design based onan allelic exchange was used, which gives rise to a thermosensitiveplasmid carrying two regions homologous to the adjacent 5 and 3′ of thewaaL gene. In order to carry out the allelic exchange by homologousrecombination, two fragments of approximately 500 bp flanking waaL inthe 3′ and 5′-regions (fragments AB and CD) were first cloned into thisplasmid. The constructed plasmid was introduced into the 3934vac strainby electroporation (the Salmonella strain carrying this plasmid is grownat 28° C. in the presence of 20 μg/mL chloramphenicol). After the strainwas transformed, several clones were selected for the integration of theplasmid into the chromosome by cultivation at 42° C. (a non-permissivetemperature for plasmid replication). After confirmation of theintegration of the vector into the chromosome by PCR, it proceeded toexcision or second recombination. In this second step, the plasmid islost having generated the desired deletion or having restored the wildcopy of the gene (the approximate ratio of these genetic events is50-50%). The second recombination is achieved by cultivation at 30° C.in the absence of antibiotic and in the presence of sucrose, since theplasmid carries the lethal sacB gene and the presence of this sugar inthe culture medium counter-selects the clones that still retain theplasmid. The confirmation of those clones in which the allelic exchangeoccurred was carried out by PCR and subsequent sequencing. It should benoted that the mutants resulting from this procedure do not carryresistance to antibiotics or traces of exogenous DNA.

In order to integrate an expression cassette into the chromosome thatwould allow the production of the fiber antigen in the SE strain, anapproach similar to that previously described was carried out. In thiscase, the plasmid contains, apart from the recombination flankingregions, an expression cassette that is integrated into the chromosome.This expression cassette was cloned into an integrative plasmid carryingthe AB and CD regions of approximately 500 bp of the Sb13 gene of theST64B prophage. The cloning was designed so that the AvA-I fiberexpression cassette is between both fragments of the defective prophagegene (the Salmonella strain carrying this plasmid is grown at 28° C. inthe presence of 20 μg/mL chloramphenicol). Once the plasmid wastransformed by electroporation, several clones were subjected to severalgrowth passages at 42° C. (non-permissive temperature of plasmidreplication—integration of the plasmid occurs thanks to the AB and CDhomologous regions adjacent to waaL). After checking by PCR that theplasmid had integrated into the chromosome, excision or secondrecombination was carried out at 28° C. in the absence of antibiotic andpresence of sucrose. As described above, this counter-selection systemallows identifying clones that have lost in the plasmid. The last stepconsists again in selecting by PCR the clones in which the insertion ofthe fiber expression cassette in the sb13 gene of the prophage ST64B hasoccurred.

The resulting strain, to which this invention refers, is a modifiedmutant strain of Salmonella enteritidis that carries a deletion of thewaaL gene, and therefore has a rough phenotype, and expresses AvA-Ifiber genes from its own chromosome.

For purposes of the present invention, Salmonella enteritidis is anabbreviation of Salmonella enterica serovar Enteritidis.

In addition, for the present invention it should be considered that PCRis the polymerase chain reaction; and oligo is the primer used in PCR.

DETAILED DESCRIPTION OF THE INVENTION

Obtaining Attenuated Salmonella Enteritidis (ASE)

Design and Construction of a System that Allows the Introduction of anyXenoantigen of Interest in the Chromosome of Salmonella enteritidis(SE).

A live vaccine or vaccine vector must be safe and effective, with agenotype and phenotype fully controlled, that avoid the risk ofreversion to virulence. In addition, the strain must maintain a balancebetween the degree of attenuation and immunogenicity, remaining in thehost organism long enough to give rise to a protective immune responseagainst homologous and/or heterologous antigens. In this case, thestrain of avian Salmonella enteritidis (ASE) generated has as its maincharacteristics a drastic attenuation in birds. In addition, the ASEstrain is unable to form biofilms and has a much lower survival in theenvironment, avoiding any risk associated with the period in which thevaccinated animals could excrete this strain. Unlike most commercialvaccines (such as 9R), its genotype and phenotype are fully controlledand do not possess antibiotic resistance genes. The safety of liveattenuated bacteria has been verified in other models such as the 9Rvaccine, whose reports scientifically ruled out a potential reversion tothe original virulent form (Okamoto, A. S., Menconi, A., Goncalves, G A.M., Rocha, T. S., Andreatti Filho, R. F., Savano, E. N., & Sesti, L.Revision to virulence evaluation of a 9R vaccine strain of Salmonellaenterica serovar gallinarum in commercial brown layers” RevistaBrasileira de Ciencia Avicola, 12(1), pp. 47-52. 2010).

Therefore, the present invention uses a strain of Salmonella enteritidis3934 (deposited in the Spanish Type Culture Collection (CECT) with theaccess number CECT 9333, to which, by genetic engineering techniques,the twelve genes encoding diguanylate cyclase enzymes, the rpoS gene andthe waaL gene have been deleted. This last mutation was carried out withthe aim of obtaining a vaccine strain of rough phenotype (Salmonellaenteritidis 3934vac DwaaL) that confers protection in breeding animals.In addition, another aspect of the present invention comprises a vaccinestrain of rough phenotype carrying an expression cassette for AvA-Ifiber genes (Salmonella enteritidis 3934vac DwaaL-fiber) which confersimmunity against inclusion body hepatitis in birds.

Generation of Rough Mutants

In order to reduce the toxicity and maintain the immunogenicity of thestrains based on the 3934vac genetic background, it proceeded to deletethe waaL gene. The mutation of this gene results in a rough phenotypebecause the lipopolysaccharide is composed only of core and lipid A,having lost the ability to synthesize the O-antigen. This mutation hasthe additional advantage of allowing the differentiation of vaccinatedand naturally colonized animals by wild strains of Salmonellaenteritidis and typhimurium.

Detailed Methodology

The methodology used is to construct an integrative vector pKO:waaL,integrate this vector pKO:waaL in a first step of recombination and thenperform the excision of the integrative vector with a second step ofrecombination (FIG. 1)

1. Construction of the Integrative Vector pKO:waaL

For the construction of the integrative plasmid pKO::waaL, two flankingfragments to the waaL gene of 520 bp (oligonucleotides A and B) and 504bp (oligonucleotides C and D), respectively, are amplified by PCR. ThePCR products are purified from gel and cloned independently in standardcloning vectors, later to be digested to NotI XhoI (fragment AB) andXhoI BgIII (fragment CD). Both digested fragments are purified from geland subcloned in the pKO plasmid, whose sequence is SEQ ID NO:1 digestedto NotI BIgII, giving rise to the vector pKO::waaL. The pKO plasmidcarries a cassette of resistance to Chloramphenicol, a thermosensitiveorigin, lacZ selection system and sacB for counter-selection (FIG. 2)

The construction pKO::waaL, whose sequence is SEQ ID NO:2, is carriedout in a strain of Escherichia coli and is verified by PCR, miniprep anddigestion.

The Oligonucleotides used for the construction of the pDEST::waaL vectorare shown in Table 1. Sequence (5′-3′). The restriction sites areunderlined.

TABLE 1 Oligonucleotides used to perform the deletion of the waaL genein Salmonella enteritidis 3934 Name of the gene Oligonucleotides waaLoligo A (SEQ ID NO: 3) oligo B (SEQ ID NO: 4) oligo C (SEQ ID NO: 5)oligo D (SEQ ID NO: 6)2. Integration of the Suicide Vector pKO::waaL (First Recombination)

Once constructed and verified the pKO::waaL plasmid (FIG. 3), this isextracted from the strain of Escherichia coli by means of miniprep andis electroporated in the S. Enteritidis 3934vac strain, where itsintegration is forced by growth at 42° C. This temperature is notpermissive for the replication of the plasmid and the integration of theplasmid occurs thanks to the homologous regions AB and CD adjacent towaaL. Several clones resulting from the incubation at 44° C., resistantto chloramphenicol, were tested by PCR with the pairs of oligos E-F andE-G to verify the integration. In the first case, the PCRs must benegative and in the second case, those clones in which the recombinationtook place in the AB fragment, the resulting PCR will be 1282 bp.

TABLE 2 Oligonucleotides used for the verification of the integration ofthe pKO::waaL plasmid Name of Oligo Sequence oligo E SEQ ID NO: 9 oligoF SEQ ID NO: 10 oligo G (hybridized in the SEQ ID NO: 11 pKO plasmid)3. Excision of the Integrative Vector pKO::waaL (Second Recombination)

In order that the second recombination event occurs and the pKO plasmidis lost, several integrated clones are grown in liquid medium at 30° C.in the absence of antibiotic. After 24 hours, 5 serial dilutions areplated on solid medium supplemented with 5% sucrose. In this medium,only those clones that have lost the plasmid through a secondrecombination grow, depending on the fragment in which it has takenplace, giving rise to the regeneration of the parental genotype or tothe total deletion of the waaL gene. Both alleles can be differentiatedby PCR with oligos E and F (sequence described above)

In the parental strain 3934vac, PCR with oligos E and F results in afragment of 2328 bp, the sequence of the amplicon from genotype 3934vacis SEQ ID NO:7, whereas in clones in which the deletion has occurred,the fragment has a size of 1106 bp, whose amplicon sequence is SEQ IDNO:8

The 3934vacDwaal or 3934vacR strain can be grown at 37° C. in theabsence of antibiotic, since it does not carry any selection cassette.

The Waa region (rfa) of Salmonella enteritidis 3934vac and the Waaregion (rfa) of Salmonella enteritidis 3934vacR can be seen in FIG. 4and FIG. 5, respectively.

The oligonucleotides used in the verification of the DwaaL mutants are:SEQ ID NO:9 and SEQ ID NO:10

Construction and Insertion of Cassette with AvA-I Fiber Genes in the3934VacR Strain

Construction and Insertion of Expression Cassette with AvA-I Fiber Genesin the Chromosome of 3934VacR.

The insertion of the genes of interest in the 3934vacR strain is carriedout at the chromosomal level. This strategy allows the expression ofheterologous genes to be stable, eliminating the problem of plasmidsegregation and the need to use antibiotics as selection markers. Thesteps are summarized in FIG. 6.

1. Construction of the Integrative Vector pKO:Sb13-Fiber

In this first phase, an integrative plasmid is constructed to integratethe expression cassette in the sb13 gene of the defective prophage ST64B(All strains of SE have it in their chromosome) (FIG. 6). Following anapproach similar to that described above, the AB (427 bp) and CD (597bp) regions are amplified with the oligo pairs H-I and J-K. The PCRproducts are purified from gel and cloned independently in standardcloning vectors, later to be digested with SmaI sphI (fragment AB) andspHI SaII (fragment CD). Both digested fragments are purified from geland subcloned in the suicide plasmid pKO digested to SmaI SaII, givingrise to the vector pKO::sb13 (FIG. 7) whose sequence is SEQ ID NO:12.

Oligos Used

Fragment AB:

oligo H (SEQ ID NO:13)

oligo I (SEQ ID NO:14)

Fragment CD:

oligo J (SEQ ID NO:15)

oligo K (SEQ ID NO:16)

Once the pKO::sb13 plasmid is constructed, the cassette is assembled,which will give rise to the expression of the fiber antigen. This stepis carried out in several phases: (i) Amplification of <Terminator (43bp)-Pr.promotor.RBS (114 bp)-clyA (915 bp)> from a synthetic plasmidwith oligonucleotides L-M, (ii) amplification of the coding region ofthe fiber antigen (1597 bp) using as template the pET28::fiber plasmidwith the pair of oligonucleotides N-O, and (iii) overlapping PCR to fuseboth fragments, with the external oligos L-O.

Oligo L (SEQ ID NO:17)

Oligo M (SEQ ID NO:18)

Oligo N (SEQ ID NO:19)

Oligo 0 (SEQ ID NO:20)

The fiber expression cassette has the sequence SEQ ID NO:21.

The resulting fragment (2,702 bp) is purified from gel and cloned byassembly of homologous ends in the vector pKO::sb13 previously digestedto spHI, obtaining the vector pKO::sb13-Fiber, whose sequence is SEQ IDNO:22.

2. Integration of the Suicide Vector pKO::Sb13-Fiber (FirstRecombination)

Once constructed and verified, the pKO::sb13-fiber plasmid, this isextracted from the Escherichia coli strain by miniprep andelectroporated into the S. enteritidis 3934vacR (or 3934vac DwaaL)strain, where its integration is forced by growth at 42° C. Thistemperature is not permissive for the replication of the plasmid and theintegration of the plasmid occurs thanks to the homologous regions ABand CD homologous to the sb13 gene. Several clones resulting fromincubation at 44° C., resistant to chloramphenicol, are tested by PCRwith the pairs of oligos P-Q and P-M to verify the integration. In thefirst case, the PCRs must be negative and in the second case, thoseclones in which the recombination took place in the AB fragment, theresulting PCR will be 1607 bp.

The oligos used were:

Oligo P (SEQ ID NO:23)

Oligo Q (SEQ ID NO:24)

Oligo M (SEQ ID NO:25)

Excision of the Integrative Vector pKO::Sb13-Fiber (SecondRecombination)

In order that the second recombination event occurs and the pKO plasmidis lost, leaving the fiber antigen expression cassette inserted into thechromosome, several integrated clones are grown in liquid medium at 30°C. in the absence of antibiotic. After 24 hours, 5 serial dilutions areplated on solid medium supplemented with 5% sucrose. Only those clonesthat have lost the plasmid through a second recombination grow in thismedium, which, depending on the fragment in which it took place, resultsin the regeneration of the parental genotype or insertion in the sb13gene of the defective prophage ST64B of the expression cassetteresponsible for the production of the fiber antigen. These latter clonescan be differentiated by PCR, using the oligos P and Q (sequencedescribed above).

In the parental strain 3934vacR, the PCR with oligos P and Q results ina fragment of 1314 bp, while in clones in which the 3934vacR::FIBER(AvA-I) strain has been produced, the integration of the fiberexpression cassette has a size of 3792 bp bp.

The sequence in the genetic background 3934vacR is SEQ ID NO:25.

The sequence sb13 in genetic background 3934vacR::FIBER (AvA-I) is SEQID NO:26.

Detection of the Expression of Fiber Proteins by ASE

For the confirmation of the heterologous expression of the fiberantigen, the SDS-PAGE and Western blot techniques are used. Theheterologous proteins are expressed by culturing the bacteria in LBmedium, then proteins are extracted from the cytoplasm, periplasm andsecreted. The samples obtained from the expression are separated bySDS-PAGE in 10% polyacrylamide gels. The proteins are transferred tonitrocellulose membranes and the proteins are detected indirectly usingIgG-HRP (antibody linked to horseradish peroxidase) as conjugate,followed by detection using DAB (3,3′ Diaminobenzidine tetrahydrochloride) as substrate.

Thus, the results obtained by Western blot validate and satisfactorilycorroborate the expression of the fiber antigen in the Salmonellaenteritidis 3934Vac strain under both 37° C. and 28° C. growthconditions. While none of the bands (A, B, and C) match exactly theestimated theoretical molecular weight of the ClyA-FiberAvAI-His fusion(85.4 kDa), band A probably matches the full size of the fusion, whilethe bands B and C are products of protein degradation. Where ClyArepresents the sequence of the ClyA gene; fiberAvAI represents the fibergene sequence of AvA-I and His represents the sequence of 6 consecutivehistidines.

Phenotypic Study of 3934 Strains and Rough Vaccine Derivatives.

In order to study the phenotypes of the different strains, they wereplated on Congo red agar, which allowed to differentiate in a simple waythe strain of Salmonella that carries the fiber gene. Unlike wildstrains, which normally produce cellulose, fimbriae and otherappendages, giving rise to the RDAR—red, dry and rough genotype, themutants generated in the present invention have a phenotype known as saw(smooth and white). This phenotype is especially noticeable when thestrains produce a truncated lipopolysaccharide, due to the mutation inwaaL gene. The differences are also significant in semisolid swimmingagar, although this phenotype may suffer variations depending on theexact composition and humidity of the culture plates (FIG. 8).

Evaluation of Recombinant Vaccine

Immunization and Challenge Tests

Preparation of the Inoculum of the Vaccine.

The inoculum is prepared from the RAvA-SE strain that adequatelyexpresses the AvA-I fiber gene. The bacterium is plated in XLD mediumand incubated for 24 hours at 37° C. Typical colonies are transferred toLB medium, culture is incubated for 18 hrs at 37° C. under stirring at250 rpm (mechanical stirrer). Bacterial cells are collected bycentrifugation at 5000 g for 10 min and serially diluted in PBSaccording to the desired concentration.

Preparation of the AvA-I Inoculum for Challenge Tests.

The AvA-I isolation used in the trial was obtained from an outbreak ofHCI (inclusion body hepatitis) from a farm in Chincha, Ica, Peru,characterized as AvA-I serotype 4 by RFLP-PCR. The isolate is inoculatedin 10-day-old SPF eggs through the chorioallantoic membrane (CAM) andincubated for 9 days, after which the livers are collected to behomogenized and the viral load is determined by PCR. The mean infectivedose in chicken embryo (IDCE 50/mL) of the inoculum is calculated usingthe formula of Reed and Muench. The homogenized material is stored at−20° C. until its use in the challenge.

Protection Test

At this point, the animal tests will allow evaluating the immunogenicityof the candidate vaccine RAvA-SE. To do this, a similar schedule isestablished as shown below. The number of groups to work is establishedaccording to the dose and route of administration. If more than onecandidate vaccine is generated, the same scheme will also be followed.Each treatment group consists of 30 animals, this number is justifiedaccording to a schedule that involves the sacrifice of animalsthroughout the experiment to evaluate colonization of the bacteria ininternal organs, where animals are understood as poultry.

A standardized vaccination schedule model was followed according to thefollowing schedule:

TABLE 3 Schedule for vaccination schedules Type of (ELISA, PCR, DaySchedule samples Laboratory Culture) 0 7 14 21 28 Date Age of 1 8 15 2229 the bird day days days days days Vaccine X 1st dose Challenge X BloodSerum LMS ELISA X X X X X Organs Liver, LBMG/LMS PCR/ X X X X X spleen,Microbiological stool, drag culture swab Other LMS: Microbiology andserology laboratory LBMG: Laboratory of molecular biology and genomics

At this point, SPF Hyline 1-day-old chickens are used, which arevaccinated according to the aforementioned scheme. After vaccination,the birds are monitored for the observation of clinical signs. Eachexperimental group is kept in separate cages. On day 21, the birds arechallenged intramuscularly with 10¹⁰ copies/mL of AvA-I serotype IV,which is equivalent to 10⁵ IDCE₅₀ (mean infective dose in chickenembryo). One of the groups are challenged with both AvA-I(intramuscularly) and with a wild strain of Salmonella enteritidis(orally, 10⁷ copies/mL). Samples are taken at regular intervals,according to the established schedule that will include feces, cloacalswabs, liver and spleen for the detection and quantification of RAvA-SE,wild SE and AvA-I. Blood samples are also considered according to theschedule for the measurement of protective antibodies. At 7 dayspost-challenge, all birds are euthanized.

Determination of Excretion and Colonization of Attenuated Salmonellaenteritidis in Chickens

At this point, the animal tests allow determining that the attenuatedSalmonella enteritidis strain (ASE) provides the characteristics underwhich it has been designed: avirulent, decreased colonization capacity,persistence and excretion, compared with a wild field strain.

The work schedule was designed specifying: treatment groups, route ofadministration, concentration of the bacteria to be used, type of sampleto be evaluated and the days of sampling. Each treatment consists of 15animals, this number includes the euthanasia of animals on the daysindicated in the schedule, to evaluate colonization of the bacteria ininternal organs. The 1-day-old birds separated by group according totreatment are inoculated with the ASE strain using a sterile 1 mLsyringe. Birds are monitored daily for clinical signs, providing foodand ad libitum water. The conditions of relative humidity and theappropriate light-dark cycles are maintained. The birds at the end ofthe experiment were euthanized by electric shock.

According to the design of the experiment, samples are sent to thelaboratory for the respective microbiological, serological and molecularanalysis.

Microbiological Assays for the Detection and Quantification ofSalmonella.

They allow determining excretion and tissue colonization of the ASEbacterium throughout the experiment, according to the established sampleshipment schedule.

Stool samples, rectal swabs, spleen and liver from the birds of thedifferent groups evaluated are analyzed throughout the experimentaccording to the established sample shipment schedule. The procedure forisolation is carried out as indicated by the flow chart (FIG. 9).

The bacterial load in the samples was determined.

LIST OF FIGURES

FIG. 1: Allelic exchange method for the construction of 3934vacR.

FIG. 2: Integrative plasmid pKO

FIG. 3: Integrative plasmid pKO::waaL

FIG. 4: Waa region (rfa) of Salmonella enteritidis 3934vac

FIG. 5: Waa region (rfa) of Salmonella enteritidis 3934vacR

FIG. 6: pKOB::sb13 Plasmid

FIG. 7: pKOB::sb13 fiber Plasmid

FIG. 8: Phenotypic differentiation of Salmonella strains on Congo redagar plates (left) and semisolid agar (right).

FIG. 9: Flow chart for the detection and microbiological quantificationof Salmonella. In addition, TSI: Triple sugar iron agar; LIA: Lysineiron agar.

FIG. 10: Western blot results of the protein of interestclyA-fiberFAdV-His.

FIG. 11: SDS-PAGE results of the protein of interest clyA-fiberFAdV-His,with positive controls A and B.

FIG. 12: Coomassie staining of the SDS-PAGE and Western Blot of theprotein extracts obtained after growth in LB medium at 37° C. or 28° C.In addition, MW: molecular weight marker, wt: Salmonella enteritidis3934, A: Salmonella enteritidis 3934Vac; A-fiber: Salmonella enteritidis3934Vac with ClyA-fiberFAdV-His fusion on the chromosome, A-pfiber:Salmonella enteritidis 3934Vac expressing the ClyA-fiberFAdV-His fusionfrom plasmid. The specific bands obtained in the western blot have beenindicated (A, B and C).

FIG. 13: Electrophoretic analysis of the PCR products reveals thepresence of clones carrying the waaL deletion. For the selection ofclones carrying the deletion, the bacterial chromosome was amplifiedusing oligos external to the construct. In addition, M=molecular weightmarker, wt=wild strain, c=colony. Expected sizes: Wt=2328 bp, ΔwaaL=1112bp

FIG. 14: Summary table of oligos used

PREFERRED EMBODIMENT OF THE INVENTION: EXAMPLES

The following examples that are provided herein serve to illustrate thenature of the present invention. These examples are included forillustrative purposes only and should not be interpreted as limitationsof the invention claimed herein.

Example 1: Expression of the Fiber Antigen in the Salmonella enteritidis3934 Vac Strain by Western Blot

Protein Extract

We plated 1 colony in 5 mL of LB culture medium for the ClyA fiber,which has the insertion in the chromosome. Incubate at 37° C. at 200 rpmovernight.

We centrifuged at 11 0000 rpm for 5 minutes and added the lysis buffer 1(Tris HCL 10 Mm, EDTA 5 mM, NaCl 50 mM)+protease inhibitor 40 μL (3g/mL)+50 μL of lysozyme (10 mg/mL), plus the SDS loading buffer (50 μLsimple buffer 2x+50 μL urea 8M+5 μL B-mercaptoethanol in 50 μL sample)per sample and all was homogenized and heated at 100° C. for 5 minutesand then placed on ice 5 minutes and from there 20 μL was taken forloading. 0.1 Amps were used for each gel by 1:30.

The band of the protein of interest clyA-fiberFAdV-His was observed inthe results of the Western blot. Markers were used; on the left theladder P7709V (175 KDa), on the right ab 116029 (245 KDa) (FIG. 10).

In addition, when two positive His+ controls are used, but the initialsA and B are by weight, A is approximately 60 kDa and B is approximately30 kDa. These results allow us concluding that the clyA-fiberFAdV-Hisfusion is expressed with a molecular weight of approx. of 85.4 kDa.(FIG. 11)

The Western blot was performed for the wild strain: Salmonellaenteritidis 3934, Δ: Salmonella enteritidis 3934Vac; Δ-fiber: Salmonellaenteritidis 3934Vac with ClyA-fiberFAdV-His fusion on the chromosome,Δ-pfiber: Salmonella enteritidis 3934Vac expressing thecly-AfiberFAdV-His fusion from plasmid.

Electrophoretic analysis of the PCR products reveals the presence ofclones carrying the waaL deletion (FIG. 13)

For the purposes of the present invention, the use of the Salmonellaenteritidis 3934vac strain in experimental models suggested that itwould work in murine and even avian model,

In our trials, we have observed that when inoculated into birds, it doesnot work well because the infection develops anyway; in order to achieveimmunity, a deletion was made in the waaL gene of Salmonella enteritidis3934vac, with which immunity was achieved. Likewise, the challenge testswith the 3934vac sb13::clyA-fiberFAdV ΔwaaL strain demonstrated that itconfers immunity against Salmonella and the avian type-I Adenoviruspreventing the appearance of inclusion body hepatitis.

Deposit of Microorganisms

The strains of SG-9R sb:ClyA-FIBER 6His, 3934 vac-rough mutant and 3934vac-Fiber rough mutant have been deposited in the Spanish Type CultureCollection (Paterna, Valencia, Spain), following the rules of theBudapest Treaty for the deposit of microorganisms for patent purposes onthe following dates and they have been assigned the following depositnumber:

Material Deposit date Access Number SG-9Rsb:ClyA-FIBER 6His Apr. 5, 2017CET 9331 3934 vac- rough mutant Apr. 5, 2017 CET 9332 3934 vac- Fiberrough Apr. 5, 2017 CET 9333 mutant

The present invention is not limited to the scope of the microorganismsdeposited in the patent, since they represent a specific illustration ofan aspect of the invention. Any microorganism or plasmid that isfunctionally equivalent to those described in the invention are includedwithin the invention.

The invention claimed is:
 1. A mutant strain of Salmonella enteritidis3934vac wherein it comprises a deletion of the waaL gene.
 2. Method forgenerating a mutant strain of Salmonella enteritidis 3934vac, wherein itcomprises: Construct an integrative vector, where for the constructionof the integrative vector, two fragments flanking the waaL gene, one of520 bp (oligonucleotides A and B) and the other of 504 bp(oligonucleotides C and D), respectively, are amplified by PCR, wherethe oligonucleotides A, B, C and D are the sequences SEQ ID NO:3, SEQ IDNO:4, SEQ ID NO:5, SEQ ID NO:6; Purify and clone the PCR productsseparately in pKOB vectors for amplification, to obtain the resultingpKOB::waaL vector for its amplification, then enzymatically digest,purify and bind in the pDESTINATION vector; Transform the resultingvector pDEST::waaL into Escherichia coli and verify by PCR, miniprep anddigestion; once constructed, the pDEST::waaL plasmid is electroporatedinto the Salmonella enteritidis 3934vac and 3934vac sb13::clyA-fiberFAdVstrains, where its integration is forced by growth at 42° C. andsubsequent excision at 28° C.; Verify the deletion of the waaL gene bydouble homologous recombination by PCR with oligos external to theconstruction (waaL E and F), where oligos E and F are SEQ ID NO:9 andSEQ ID NO:10; and Adding said modified mutant strain of Salmonellaenteritidis 3934vac to a composition to make a vaccine comprising saidmodified mutant strain of Salmonella enteritidis 3934vac with a deletionof the waaL gene.
 3. A mutant strain of Salmonella enteritidis 3934vacwherein it comprises the complete sequence of the AvA-I fiber gene and adeletion of the waaL gene.
 4. Method for generating a mutant strain ofSalmonella enteritidis 3934vac wherein it comprises: Have a strain ofSalmonella enteritidis 3934vac; Insert a selection cassette by means ofa first step of homologous recombination, wherein the cassette containssequences complementary to the prophage ST64B in its flanking regions,in addition to the gene for resistance to antibiotic; where thebacterium is electroporated in the presence of the linear DNA carryingthe selection cassette in the first step of homologous recombination;Select the transformant colonies in LB medium containing the antibioticafter the first step of homologous recombination; Confirm the presenceof the cassette in the chromosome of Salmonella enteritidis 3934 by PCRwith the oligos of sequences SEQ ID NO:23, SEQ ID NO:24 and SEQ IDNO:257; Insert a ClyA-fiberFAdV-His expression cassette by a second stepof homologous recombination, where the expression cassette replaces theselection cassette, where the expression cassette comprises the sequenceSEQ ID NO:21; and Select the strains of Salmonella enteritidis 3934vacsb13::clyA-fiberFAdV by verifying the product of the expression of thesequence SEQ ID NO:21 by Western Blot and SDS-PAGE.
 5. Method accordingto claim 4, wherein it also comprises: Construct an integrative vector,where two fragments flanking the waaL gene, one of 520 bp gene(oligonucleotides A and B of sequences SEQ ID NO:3 and SEQ ID NO:4,respectively) and other of 504 bp (oligonucleotides C and D of sequencesSEQ ID NO:5 and SEQ ID NO:6, respectively) are amplified by PCR for theconstruction of the integrative vector; Purify and clone the PCRproducts separately in pKOB vectors for amplification, to obtain theresulting vector pKOB::DwaaLR1 for its amplification, then enzymaticallydigest, purify and bind in the pDESTINATION vector; Transform theresulting vector pDEST::waaL in Escherichia coli and verify by PCR,miniprep and digestion; once constructed, the pDEST::waaL plasmid iselectroporated in the Salmonella enteritidis 3934vac and 3934vacsb13::clyA-fiberFAdV strains, wherein its integration is forced bygrowth at 42° C. and subsequent excision at 28° C.; and Verify by PCRwith oligos SEQ ID NO:9 and SEQ ID NO:10, external to the construction,the deletion of the waaL gene by double homologous recombination. 6.Recombinant avian vaccine of Salmonella enteritidis wherein it comprisesa modified mutant strain of Salmonella enteritidis 3934vac with asequence of the AvA-I fiber gene and a deletion of the waaL gene.
 7. Amutant strain of Salmonella enteritidis 3934vac wherein it comprises anexpression cassette SEQ ID NO:21 and the deletion of waaL that isverified by the presence of the sequence SEQ ID NO:8.
 8. A mutant strainof Salmonella enteritidis 3934vac wherein it comprises a sequence SEQ IDNO:26 and SEQ ID NO:8.